In general, the following conditions are considered as essential for establishment of pregnancy:
(1) The existence of sufficient number of sperms having sufficient motility and fertility in an ejaculated seminal fluid; PA1 (2) Normal growth and maturation of ovarian follicles and ova, and discharging of an ovum in ovulation which has fertilizability and cleaving ability; PA1 (3) The form and function of an oviduct maintained normally; and PA1 (4) The form and function of a uterus being normal for embryo implantation and maintenance thereof.
In the present situation, however, at least one out of ten married couples is estimated as being unable to satisfy one or more above-described conditions, namely, is infertile. The cases can be broadly classified into 40% of the cases where the causes can be attributed to female factors; 25% of the cases where the causes can be attributed to male factors; 25% of the cases where the causes can be attributed to both female and male factors; and 10% of cases where the causes cannot be clarified.
In respect of such causes, incurable cases as described below are called "absolute infertility": No ova to be discharged are present; the uterus has been extirpated; the husband is azoospermia; or the like. Hitherto, although the case in which both oviducts had been extirpated was regarded as absolute infertility, pregnancy and birth even in such cases have become possible by means of in vitro fertilization (IVF) or embryo transfer (ET). According to development of such an in vitro fertilization or embryo transfer method, pregnancy in cases such as atretic oviduct or oligozoospermia, in which pregnancy had been impossible, has become possible. The ratio of pregnant cases by in vitro fertilization to the total number of in vitro fertilization operations is still as low as approximately 20 to 30%, and the ratio of the cases resulting in birth is only 5 to 10%. Concerning such low ratios, implantation failure in returning fertilized ova to the uterus is considered as a cause. In addition, implantation disability is pointed out as possibly occupying a high percentage of the causes for unaccountable infertility or so called functional infertility.
Although artificial insemination is also frequently performed in animals other than human beings, the conception rate and birth rate therein are not necessarily satisfactory. In particular, when such operations are performed for preservation and reproduction of rare animals, such low rates are problems since there exists a temporal limitation due to the high ages of the remaining animals or the like. Similar to cases of human beings, it is considered that such low rates may be attributed to a failure in sufficiently satisfying the above-described conditions in the steps for establishing pregnancy.
Through a series of developmental stages, an ovum is formed from an oocyte which has been derived from an oogonium. In mammals, an oogonium stops dividing just before or just after birth, and transforms into an oocyte which is a meiotic-type cell. In the latter stage of the prophase of the first meiosis, the oocyte comes into a stationary phase, and changes in its chromosomes do not progress (interphase of the first meiosis). At this time, the nucleic volume of the oocyte increases, and such an oocyte is called a "protoblast" (or "germinal vesicle"). The oocyte, then, restarts meiosis in response to gonadotropin secreted from the pituitary gland. In many mammals, the meiosis is restarted in an ovarian follicle before ovulation; the protoblast is then broken (germinal vesicle breakdown); a first polar body is released through the metaphase, the anaphase and telophase of the first meiosis; and the chromosomes stop changing in the metaphase of the second meiosis (interphase of the second meiosis). According to fertilization, the second meiosis restarts, a second polar body is released, and the meiosis is thus completed ["Jikken Seishoku-Seirigaku no Tenkai (Development of Experimental Reproductive Physiology)", vols. 11 to 16 edited by Yosisuke Suzuki, and published by Soft Science Co., Ltd, 1982]. Investigations on ovum maturation have been conducted principally using in vitro culture systems while employing the following three methods depending on the purposes of experiments.
(1) Culturing oocytes harvested from ovarian follicles under several conditions. This method is employed for analysis of factors influential upon ovum maturation.
(2) Culturing oocytes together with theca cells, or culturing oocytes in a conditioned medium based on ovarian follicles. This method is employed for analyzing influences by theca cells.
(3) So called "organ culture" in which ovarian follicles including oocytes are externally cultured. This method is employed for analysis of factors influential upon ovum maturation passing through or with intervention of thecae.
As the first step for conception, an adequate maturation process for a vital ovum is essential. The term "ootid" means an ovum which can release a second polar body in response to fertilization, and thereby complete fertilization as well as meiosis (namely, having fertilizability), and which can develop in response to fertilization stimulation (namely, having developmental potency).
The latter step for conception includes the activity of a fertilized ovum traveling through an oviduct toward the inner surface of a uterus for implantation. When a disability concerning either of the above-described steps is present on the female side, conception cannot be achieved or can be achieved only with a markedly low degree of probability though depending on the degree of the disability. Implantation is understood as an adhesion phenomenon between a fertilized ovum and endometrium. Upon implantation, endometrium as a receiver for a fertilized ovum should sufficiently thicken, and the interstitial tissue should be edematized to be soft. It was reported that implantation of a fertilized ovum rarely occurs if the interstitial tissue does not swell in the ovulation period. In the above-described infertility cases in human beings, several therapeutic treatments are performed depending on the causes thereof. For example, for oligozoospermia or azoospermia, sperms manually obtained are injected on an ovulation day into the uterus lumen or the oviduct in an attempt to achieve natural fertilization in the oviduct. Nowadays, a just ejaculated seminal fluid is not used for fear of bacterial infection, and therefore, washed and condensed sperms are used. In many cases, however, a few to tens of attempts at insemination are required until conception is achieved, and unsuccessful implantation is considered as one reason for such requirement.
Further, in vitro fertilization and embryo transfer are performed in cases such as atretic oviduct or oligozoospermia where other therapeutical treatments are not effective, wherein ova taken out of a body are inseminated with sperms, and after fertilization, divided and grown embryos are transvaginally transferred on the inner surface of a uterus. In practice, the operation comprises the following significant steps: (1) Induction of superovulation, and determination of a ovum collection period; (2) an operation for ovum collection; (3) supplemental culturing for ovum maturation; (4) collection of satisfactory sperms and capacitation; (5) in vitro fertilization; (6) culturing of the fertilized ova; (7) embryo transfer; and (8) luteal phase management. Without completion of these steps, pregnancy cannot be expected to occur. Since the endocrine system is not physiologically natural in such an estrus cycle with superovulation induction, hormonotherapy as an luteal phase management, such as administration of progesterone or the like for approximately one week after the embryo transfer, is performed for luteinization, namely thickening of endometrium and softening of the interstitial tissue. Nevertheless, the pregnancy rate is unsatisfactory, as described above, and therefore, there are demands for a further improved method.
As a modification of in vitro fertilization/embryo transfer, a related art generically called "assisted reproductive technology" has been developed. This technology includes intraoviduct zygote transplantation in which in vitro fertilization is performed, and then fertilized ova, either before division or after division and growth, are transplanted into an oviduct; and intraoviduct gametes transplantation in which collected ova in an early phase are transplanted together with sperms into an oviduct. Although these are methods which provide conditions closer to the physiologically natural process of pregnant, problems concerning implantation of a fertilized ovum has not yet been solved, and there is room for improvement.
Meanwhile, inflammation reactions as bioprotective reactions are observed not only in bacterial or viral infections and injuries, but also in tissue damage due to autoimmune reactions. At this time, specific peripheral leukocytes infiltrate into the inflammatory region. For leukocyte migration on such occasions, chemokines (chemotactic factors) play an important role. Interleukin-8 is one of such chemokines, and its excessive production is considered as a cause of several inflammatory diseases.
Interleukin-8 has been reported to be produced from several types of cells such as fibroblasts and several tumor cells as well as hemocytes such as monocytes, macrophages, and lymphocytes in response to stimulation by IL-1, TNF, LPS, or the like. Accordingly, interleukin-8 is deduced to be an important mediator for acute inflammation, and abnormal production is considered relating to some diseases. Examples of such diseases include rheumatoid arthritis, gouty arthritis, asthma, septicemia, immunological vasculitis, hepatitis, and pyelonephritis. The activity of interleukin-8 in such diseases has, however, been known only at a basic science, and the mechanism of how it relates to progress in pathologic processes of such diseases has not yet been clarified. Interleukin-8 is a chemokine which has the functions of causing chemotaxis of neutrophils and lymphocytes, and activating neutrophils and other mononuclear leukocyte or the like. The term "chemokine" means a bioactive substance (or cytokine) which has an activity of causing chemotaxis. Interleukin-8 is a protein comprising 69 to 77 amino acids. The number of amino acids alters depending on the situation since the N terminus processed with intra- or extracellular enzymes. At first, interleukin-8 was reported in the name of MDNCF (K. Matsushima, et al., J. Exp. Med., 167, 1883, 1988), after that, it was also found by other researchers and named differently, and the name was unified as "interleukin-8" (C. G. Larsen, et al., Science, 243, 1464, 1989). Interleukin-8 belongs to the CXC chemokine family, the members of which have similar amino acid numbers and similar cysteine residue configurations. "CXC chemokine family" is a generic term for a group of low-molecular weight proteins which mutually have amino acid homologies of approximately 30%, and the same four cysteine residue positions. Structural analogues of interleukin-8 such as .gamma.IP-10, GRO (.alpha., .beta., .gamma.), PF-4, and NAP-10 are known as proteins belonging to this family other than interleukin-8 (N. Mukaida, et al., Microbiol. Immunol., 36, 773, 1992). Proteins of the CXC chemokine family are characterized by having a N terminus amino acid sequence in which two cysteine residues are bonded with an intervening amino acid residue. "CXC chemokine" is also called ".beta. chemokine" (J. J. Oppenheim, et al., Annual. Rev. Immunol., 9, 617, 1991).
Monocyte chemotactic and activating factors (hereinafter referred to as MCAF) have been reported to be produced from several types of cells such as fibroblasts, endothelial cells, smooth muscle cells and several tumor cells as well as hemocytes such as monocytes, macrophages, and lymphocytes in response to stimulation by IL-1, TNF, IFN-.gamma., LPS, or the like. Additionally, the monocyte chemotactic and activating factor is also called MCP-1 (monocyte chemoattractant protein-1) or GDCF (glioma-derived monocyte chemotactic factor), and is a protein comprising 76 amino acids and having 4 cysteine residues. Reports have been made concerning identification and gene cloning of MCAF, MCP-1 or GDCF (K. Matsushima, et al., J. Exp. Med., 169, 1485, 1989; Y. Furutani, et al., Biochem. Biophys. Res. Commun., 159, 249, 1989; E. R. Robinson, et al., Proc. Natl. Acad. Sci. USA, 86, 1850, 1989; and T. Yoshimura, et al., FEBS Letters, 244, 487, 1989). Hereinafter, in the present invention, "MCAF" is used as a generic term also including MCP-1 and GDCF.
MCAF belongs to the CC chemokine family, the members of which have similar amino acid numbers and similar cysteine residue configurations. "CC chemokine family" is a generic term for a group of low-molecular weight proteins which mutually have amino acid homologies of approximately 30%, and the same four cysteine residue positions. Structural analogues of MCAF such as RANTES, LD78, ACT2, I-309, MCP-2 and MCP-3 in human beings, or JE, MIP-1 .alpha., MIP-1 .beta., and TCA-3 in mice are known as proteins belonging to this family other than MCAF (N. Mukaida, et al., Microbiol. Immunol., 36, 773-789, 1992). Proteins of the CC chemokine family are characterized by having a N terminus amino acid sequence in which two cysteine residues are bonded in series. "CC chemokine" is also called ".beta. chemokine" (J. J. Oppenheim, et al., Annual. Rev. Immunol., 9, 617, 1991).
The present inventors have reported that administration of an interleukin-8 suppository to a rabbit can cause cervical ripening (El Maradny, et al., Am. J. Obstet. Gynecol., 171, 77, 1994). Cervical ripening is essential for parturition, and cervical ripening incompetency is the principal cause of today's dystocia. By local administration of interleukin-8, inflammatory cells such as neutrophils migrate into the cervix to release collagenase, elastase or the like which degrades collagen in the interstitial tissue, increase the content of water in the interstitial tissue, and thus cervical ripening takes place. However, effects of interleukin-8 or MCAF upon the steps of oogenesis, ovum maturation, fertilization, and achievement of conception have not yet been known.
The following are known as examples of means for improving conception rate: Inductive substances for resumption of the first meiosis such as cyclic AMP, calcium ions, prostaglandins, cholera toxins, and forskolin; follicle stimulating hormone (FSH) or the like for attempting follicular maturation; estrogen or the like for attempting to provide developmental potency for fertilized ova; and luteal phase management using a steroid agent or the like for attempting to achieve implantation of fertilized ova.
According to these means, however, a satisfactory pregnancy rate may not be achieved, or some agents cannot actually be used or can be used with limitations due to side effects inherent in the uses thereof.